Project Summary CytoImage Dx in collaboration with OHSU will establish a commercial platform technology that offers unprecedented molecular-level sensitivity, increased multiplexing capability, and minimized patient sample requirement, thereby providing functional profiling of increased numbers of drug combinations with improved single cell granularity. This will help accelerate target pathway identification of synergistic compounds most effective in killing cancerous cells while minimizing drug-resistant minor cell subpopulations. Current flow cytometry platforms query cell identity but are sample-limited to 1-2 drug combination agents and do not offer reliable measures of key, clinically-relevant proteins (e.g. phosphoproteins) that are challenging to detect in single cells, due to their low basal levels. Thus, many drug targets and patient samples cannot be evaluated and the absence of protein detection cannot be reliably interpreted as a negative result. OHSU has built an imaging platform addressing these technical bottlenecks that is currently used in NCI-sponsored projects to screen patients enrolled in Beat AML, the world?s most comprehensive AML cohort to date that is nominating imuno-onc/small molecule drug combinations for acute myeloid leukemia to clinical trials. We have shown that the platform identifies small molecule/immune checkpoint inhibitor combinations that more effectively induce leukemia cell kill and immune cell proliferation. The platform requires x100-fold less patient sample size, making feasible the evaluation of expanded drug combinations and functional characterization of targeted pathways and individual cells/cell subtypes (e.g. T cell, leukemic cell). Proprietary super-localization algorithms discriminate discrete protein complexes tagged with antibody-fluorophore probes, offering up to x100 times increased signal to noise over flow cytometry and fluorescence microscopy (Jacob et al, Sci Reports 2016; 2 patents issued, 1 pending), enabling reliable detection of drug-resistant progenitor cells. In this SBIR Phase I application, we propose to benchmark platform performance to obtain CLIA-forward specs necessary for commercialization, to enhance the multiplexing capability beyond current state-of-the-art, and to profile targeted pathways and cell subpopulations responding to small-molecule/immune checkpoint inhibitor combinations that produce the most effective tumor cell kill/T-cell proliferation, while minimizing residual phosphoactive, drug-resistant cells. This would allow Phase II focus in testing in expanded patient cohorts. The collaborative team consists of innovators of the single cell imaging platform, clinician- scientists leading efforts to identify leukemia-targeted therapeutics for clinical deployment, and an executive-in-residence with a track record of fostering successful start-ups. CytoImage Dx has an option agreement to exclusively license the OHSU platform technology. Letters of support attest to the innovation, clinical significance, and institutional support of the technology for commercial spin-out.